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Ruva Is a Sliding Collar That Protects Holliday Junctions from Unwinding While Promoting Branch Migration

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Ruva Is a Sliding Collar That Protects Holliday Junctions from Unwinding While Promoting Branch Migration doi:10.1016/j.jmb.2005.10.075 J. Mol. Biol. (2006) 355, 473–490 RuvA is a Sliding Collar that Protects Holliday Junctions from Unwinding while Promoting Branch Migration Daniel L. Kaplan1* and Mike O’Donnell1,2 1Rockefeller University, The RuvAB proteins catalyze branch migration of Holliday junctions Laboratory of DNA Replication during DNA recombination in Escherichia coli. RuvA binds tightly to the New York, NY 10021, USA Holliday junction, and then recruits two RuvB pumps to power branch migration. Previous investigations have studied RuvA in conjunction with 2Howard Hughes Medical its cellular partner RuvB. The replication fork helicase DnaB catalyzes Institute, Laboratory of DNA branch migration like RuvB but, unlike RuvB, is not dependent on RuvA Replication, New York, NY for activity. In this study, we specifically analyze the function of RuvA by 10021, USA studying RuvA in conjunction with DnaB, a DNA pump that does not work with RuvA in the cell. Thus, we use DnaB as a tool to dissect RuvA function from RuvB. We find that RuvA does not inhibit DnaB-catalyzed branch migration of a homologous junction, even at high concentrations of RuvA. Hence, specific protein–protein interaction is not required for RuvA mobilization during branch migration, in contrast to previous proposals. However, low concentrations of RuvA block DnaB unwinding at a Holliday junction. RuvA even blocks DnaB-catalyzed unwinding when two DnaB rings are acting in concert on opposite sides of the junction. These findings indicate that RuvA is intrinsically mobile at a Holliday junction when the DNA is undergoing branch migration, but RuvA is immobile at the same junction during DNA unwinding. We present evidence that suggests that RuvA can slide along a Holliday junction structure during DnaB-catalyzed branch migration, but not during unwinding. Thus, RuvA may act as a sliding collar at Holliday junctions, promoting DNA branch migration activity while blocking other DNA remodeling activities. Finally, we show that RuvA is less mobile at a heterologous junction compared to a homologous junction, as two opposing DnaB pumps are required to mobilize RuvA over heterologous DNA. q 2005 Elsevier Ltd. All rights reserved. Keywords: DNA replication; DNA recombination; Holliday junction; branch *Corresponding author migration; RuvA Introduction which is processed by the RuvABC proteins.2 The RuvA protein initially binds the Holliday junction, DNA recombination functions in Escherichia coli and then recruits RuvB protein rings to opposite torepairdamagedDNAandrescuestalled sides of the junction.3,4 RuvB is a molecular motor replication forks.1 During recombination, a DNA that uses the energy derived from ATP binding and strand is paired with its homolog from a different hydrolysis to drive unidirectional movement of the duplex in a reaction catalyzed by RecA working in four-way junction.5 The RuvAB complex then concert with other proteins.1 This creates a four- recruits RuvC to the junction.6 RuvC is a nuclease way DNA structure, also called a Holliday junction, that cleaves the Holliday junction, thereby resolving it into two duplex DNAs.7 RuvAB has been studied by biochemical and Present address: D. L. Kaplan, Vanderbilt University, Department of Biological Sciences, Nashville, TN 37232, structural techniques. RuvA binds as a tetramer or USA. octamer to a Holliday junction. The RuvA protein Abbreviation used: ssDNA, single-stranded DNA. has acidic pins that inhibit binding to double- E-mail address of the corresponding author: stranded DNA, thereby targeting the protein to 8–11 [email protected] Holliday junctions. RuvB is a hexameric ring 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. 474 RuvA Promotes Branch Migration protein with a central channel wide enough to mobilizes RuvA using only RuvA and RuvB, since encircle double-stranded DNA.11–13 RuvB alone the activities of these two proteins are dependent does not bind Holliday junction DNA under upon one another.2 Unlike RuvB, DnaB does not physiological conditions, but after RuvA binds the require RuvA for activation. Thus, we investigated Holliday junction, RuvA facilitates the assembly of how RuvA moves at a Holliday junction by using two RuvB rings onto opposite sides of the RuvA- DnaB in conjunction with RuvA. In this study, DnaB junction (Figure 4(a)).14–16 The two RuvB rings are is used as a tool to study how RuvA functions, since thought to function in concert as DNA pumps that these two proteins do not function together in vivo. drive branch migration of the Holliday junction.15 There is an additional advantage to using DnaB DnaB functions in DNA replication and is a with RuvA to study RuvA function. RuvB rings member of the class of ring-shaped helicases.17–19 bind to opposite arms of a RuvA-bound Holliday DnaB is the primary replicative helicase of E. coli, junction in either of two orientations (Figure 4(a)). and unwinds the parental duplex to provide single- Thus, it is difficult to target RuvB loading to a stranded DNA (ssDNA) for the replicative poly- particular junction arm. In contrast, DnaB loads merases.20 The DnaB hexamer encircles ssDNA onto junction-arm DNA only if a 50single-strand while translocating along it, pumping the strand extension (50 tail) is attached to a particular junction through the central channel.21–24 Upon encounte- arm. Thus, unlike RuvB, one DnaB hexamer can be ring a forked duplex structure, the second DNA loaded onto a particular junction arm by specifically strand cannot fit into the central channel of DnaB, adding a 50 loading tail. and therefore the continued advance of DnaB along We find that RuvA binds tightly to Holliday the original strand forces its separation from the junction DNA and blocks DnaB-catalyzed unwin- second DNA strand.24,25 It is thought that DnaB, ding of a Holliday junction. Unwinding activity is like other hexameric helicases, may act at the blocked even when two DnaB hexamers act in replication fork to unwind parental DNA in this concert. However, RuvA does not block DnaB- manner.26–28 catalyzed branch migration of a homologous Holli- The DnaB hexamer can also operate in a mode in day junction. Hence, RuvA does not need specific which it encircles both strands of duplex DNA. In protein activation by RuvB to mobilize in the this mode, the DnaB does not unwind the DNA.24 direction of branch migration. We present evidence However, DnaB actively translocates along the that suggests that RuvA slides along the Holliday duplex while encircling two DNA strands as it junction during DNA branch migration, but not powers branch migration of Holliday junctions.29 during DNA unwinding. Interestingly, two DnaB Although this reaction is very efficient in vitro, the pumps are needed to power migration of RuvA in vivo role for DnaB-catalyzed branch migration is over heterologous DNA, an action that fits nicely unclear. In summary, DnaB unwinds DNA when with the physiological architecture of two RuvB encircling one DNA strand, and drives DNA branch pumps straddling the RuvA protein at a Holliday migration while encircling two DNA strands. junction. DnaB and RuvB have several mechanistic features in common. For example, DnaB and RuvB are both hexameric ring proteins that encircle Results DNA.2 Furthermore, both DnaB and RuvB utilize ATP binding and hydrolysis to unwind DNA with Homologous and heterologous Holliday 50 to 30 polarity.24,30–32 DnaB and RuvB also displace junction DNA substrates used in this study proteins bound to DNA,29,33 and they both drive branch migration of Holliday junctions.15,29 Finally, Holliday junctions in the cell are usually homo- two DnaB rings can bind to opposite sides of a logous, as RecA normally pairs DNA strands of the Holliday junction, and work in concert to drive same sequence to create the junction. By homolo- branch migration of an extended heterologous gous, we mean that the DNA arms contain junction, like RuvB.34–36 complementary sequences before and after branch Biochemical studies of RuvA in the past have migration. Figure 1(a) shows a homologous Holli- been performed with RuvA in conjunction with its day junction substrate used in this study. Note that cellular partner, RuvB. The biochemical action of the substrate is not completely homologous, other- the RuvAB complex is thus well studied. However, wise the Holliday junction is unstable and can some intrinsic properties of RuvA during its action migrate spontaneously. Thus, the homologous are unclear, as it is most often studied in conjunction substrate used here bears a small degree of with RuvB. This leaves unanswered a number of heterology (5 bp) to stabilize the structure and questions of how RuvA functions. For example, render it amenable to experimentation. In the how does RuvA bind tightly to Holliday junctions, reaction shown in Figure 1(a), the Holliday junction yet become activated to move during branch branch-point migrates a distance of 45 bp, of which migration? Previous proposals suggest that RuvB 40 are complementary, while five are non-comple- must mobilize RuvA bound to a Holliday junction, mentary. and that this action is mediated via specific protein– Heterologous junctions contain DNA sequence protein interaction between RuvB and RuvA.10 that is non-complementary, and the DNA arm will However, it is difficult to study the process that therefore become unpaired after branch migration RuvA Promotes Branch Migration 475 Figure 1. Homologous and heterologous Holliday junction DNA substrates used in this study. The substrates shown are used in this study to assess RuvA mobility at (a) a homologous and (b) heterologous Holliday junction. (a) This homologous substrate is used in Figure 2(a). The 1-2 and 3-4 duplexes are 45 bp in length, and the 1-4 and 2-3 duplexes are 25 bp in length.
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